Classical and molecular studies enhancing the detection and control of avian leukosis virus
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The study is to enlighten the poultry industry in regards to diagnostics and possible therapeutic agents that can be adopted for avian leukosis virus (ALV) infected flocks. The ability to substitute primary cell culture with a continuous cell line is investigated. Secondly, the sensitivity and superiority of a developed nested reverse transcriptase-polymerase chain reaction (RT-PCR) in comparison to virus isolation coupled with antigen capture-enzyme linked immunosorbent assay (ac-ELISA) in the identification and detection of avian leukosis virus were evaluated and discussed. Lastly, the design and evaluation of a virtually un-degradable morpholino (MO) antisense oligo to potentially inhibit avian leukosis virus subgroup J (ALV-J) is assessed. The permissibility of the DF-1 cell line and C/E secondary chicken embryo fibroblasts (CEF) to propagate and subsequently detect ALV-J by ac-ELISA was similar when expressed in terms of percent positive and although not significantly different (Chi-square test, p>0.05) virus propagation in the DF-1 cell line allowed for the identification of more positive plasma samples at both (sample to positive) S/P ratios of 0.2 and 0.1. A 20% - 25% loss of sensitivity and significant difference at a 0.2 S/P ratio (p<0.05) was observed for ALV-J detection for DF-1 viral propagation followed by ac-ELISA when compared to nested RT-PCR. Seven virus isolation negative plasma samples which were positive for ALV-J viral RNA by nested RT-PCR were analyzed and several amino acid residues changes within the spacer region (SP), were identified. Some of these same amino acid changes identified in the ALV-J genomic RNA from the VI- plasma samples have been shown to abrogate infectivity of ALV molecular infectious clones. In addition, ALV-J antibody positive samples were detected between 2-3 log10 dilutions lower in cell culture when compared to detection by nested RT-PCR, while antibody negative samples exhibited similar detection limits for both assays. A significant reduction (p < 0.05) in the production of p27 was observed for only the MO targeted to the ADOL-7501 primer binding site (PBS). Real time quantitative LightCycler ® RT-PCR and an endpoint dilution assays were confirmed a one log reduction in viral RNA copies and infectious units detected when compared to the groups treated with a non-specific MO.